1. Field of the Invention
The present invention is broadly concerned with a multiple-peptide channel assembly which provides transport of anions through epithelial cell membranes wherein the preferred peptides have from about 18-30 amino acid residues and are soluble in water to a level of at least 10 mM; such channel assemblies can be used in the treatment of diseases such as cystic fibrosis (CF) and adult polycystic kidney disease (APKD). More particularly, the invention pertains to such channel assembly forming peptides, and corresponding methods of use, wherein the peptides are a segment of a native (i.e., naturally occurring) channel protein and have their water solubilities enhanced by modification of the C- or N-ends thereof modified with a plurality of polar amino acid residues such as lysine.
2. Description of Prior Art
Introduction. A major problem in CF is the inability of airway epithelia to secrete fluid. The resulting changes in the composition of the mucous coating the airway epithelia result in infection and subsequent inflammation, scarring, and eventual pulmonary destruction. The basis of the problem is the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane of the epithelial cells. This leads to an increase in the absorption of salt and water and an inability to respond to appropriate stimuli by secreting chloride and water. CFTR is a chloride channel; in addition it down-regulates sodium channels and up-regulates another population of chloride channels, the outwardly rectifying chloride channel (ORCC) (1). These properties of CFTR enable the airway cells to secrete chloride and this drives the secretion of sodium and water.
Two groups reported that a synthetic-23-residue .alpha.-helical peptide forms anion-selective channels in phospholipid bilayers. The peptide has the amino acid sequence of the putative transmembrane segment M2 of the strychnine-binding a subunit of the inhibitory glycine receptor and is named M2GlyR (FIG. 1; Sequence ID No. 1) (2, 3).
The origin and properties of M2GlyR. The glycine receptor is a membrane protein present in post-synaptic membranes. Binding of glycine activates a Cl.sup.- conducting channel, leading to hyperpolarization of the membrane and inhibition of the synapse. The receptor consists of two major glyco-polypeptides, an .alpha.subunit of 48 kd and a .beta. subunit of 58 kd, and a receptor-associated cytoplasmic protein of 93 kd (4). Strychnine, an antagonist of the glycine receptor, binds only to the a subunit. Messenger RNA corresponding to this subunit leads to the expression of functional, glycine-activated, Cl.sup.- channels upon injection into Xenopus oocytes (5-7).
The glycine receptor channel in cultures of embryonic mouse spinal cord is selective for monovalent anions, with conductances of 27 and 46 pS in 145 mM Cl.sup.- solution (8,9). Pharmacological studies suggested the presence of two sequentially occupied anion binding sites in the channel. These sites are considered to be the functional correlates of the positively charged amino acids bordering the M2 segment of the a subunits (8). This finding led to the development of the synthetic peptide with the sequence of the M2 segment of the glycine receptor.
Electrical recordings from phospholipid bilayers containing M2GlyR showed single-channel conductances of 25 pS and 49 pS in symmetric 0.5 M KCl with channel open lifetimes in the millisecond range. Single channel events occurred in 0.5 M N-methyl-D-glucamine HCl but not in sodium gluconate, indicating that the channel is anion selective. A transference number for anions of 0.85 was calculated from reversal potential measurements under a 5-fold KCl concentration gradient (10).
After insertion into the lipid bilayers the monomeric peptides self-assemble to form conductive oligomers of different amplitudes. To gain control over the aggregate number, four identical M2GlyR peptide units were tethered to a 9-amino acid carrier template to form a four-helix bundle protein. This tetramer, incorporated into lipid bilayers, formed channels of uniform unitary conductance of 25 pS. The 49 pS conductance described above is presumed to be due to the presence of a pentamer (10).
The tetrameric channel was blocked by the Cl.sup.- channel blockers 9-anthracene carboxylic acid (9-AC) and niflumic acid (NFA). It was not blocked by QX-222, an analogue of lidocaine and a blocker of cation-selective channels. Strychnine, an antagonist of the glycine receptor, does not block the channel-forming tetramer. Strychnine is presumed to bind to the ligand-binding domain of the receptor exposed to the extracellular surface but not to the channel domain (10).
Structure of channel forming peptides. While great strides have been made in the area of channel function and regulation, using the intact protein or in some cases purified channel proteins reconstituted into model membranes, many aspects of channel function remain unresolved. A key element, an atomic coordinate three dimensional structure of any mammalian channel protein, is not yet available. These data sets are crucial for the unambiguous assignment of the contributions individual segments make to the overall geometry of the intact protein and would contribute greatly to the understanding of the dynamics involved in channel function. The lack of structural data is due in part to the large size of these molecules but more likely due to the difficulty encountered in crystallizing membrane proteins (11). Solution NMR structural data are lacking primarily due to the large size and hence slow tumbling of the proteins embedded in lipid micelles.
Structural data does exist for the related class of channel forming peptides (CFPs). These channels are much smaller in size and contain only a ring of short peptide chains organized around the central ion conducting pore in the lipid bilayer. These channels are unique in that they assemble by the oligomerization of a single peptide. These structures are models for studying the structure and function of the various regulated channels that occur in nature. This class of CFPs includes: the .alpha.-aminoisobutyric acid-containing channels such as alamethicin and zervamicin, and a number of toxins and venoms such as melittin, cecropins, mast cell degranulating peptides, and the defensins. Melittin is somewhat of a special case because it forms channels only at low concentrations; at higher concentrations it acts as a lytic agent (12). In some cases CFPs assemble spontaneously upon insertion into the bilayer while in the remaining cases the assembly requires an electrical potential across the membrane (V.sub.m).
The structure of the channels arising from the assembly of these peptides vary from trimers to hexadecamers associated in the form of helical bundles or .beta.-barrels. The most widely accepted model which is in accord with the model for channel proteins has the helices arranged with their dipoles all pointing in the same direction (parallel) (13,14). Since CFP channels, unlike authentic channel proteins, are not generated from the association of large protein subunits, alternative stabilization schemes must be invoked to account for the presence of this higher energy arrangement of parallel segments. These could include aligning the dipoles in response to the presence of the membrane potential and/or an increase in the favorable inter-molecular interactions promoted by the parallel assembly. Most CFPs form multiple size bundles of parallel segments (e.g., n=4, 5, 6) that can spontaneously increase or decrease in size upon the addition or deletion of a peptide monomer from the channel assembly. These Observations imply that enough information is contained in a single channel forming polypeptide to drive the correct folding, assembly, and activity of these channels.
The activity of these assembled molecules, the opening and closing of the channels on the millisecond time scale, has been ascribed to numerous effects. Three different helical motions have been implicated (15): the bending and twisting of the helices, rigid-body fluctuations of the entire assembled structure with the lipid bilayer, and rotational motions of the polypeptide around its helical axis. Another hypothesis suggests that channel activity is a consequence of a conformational change that is transmitted along the helical axis (16,17). Others suggest that the movement of individual amino acid side-chains could provide this function (18), and one group contends that an electron transfer could disrupt a hydrogen bonding of four tyrosines in K.sup.+ channels (19).
Fluorescence (15,20-22), Fourier transform infrared spectroscopy (FTIR) (23-25), and circular dichroism (CD) measured in organic solvents, phospholipid micelles, liposomes, or oriented phospholipid bilayers (15,20,21,24-34) have been successfully used to probe the solution and membrane-bound conformations of these peptides. Computer modeling studies have been performed to estimate the energetics of moving a charged ion across a lipid bilayer through a pore generated by a bundle of transmembrane helices (35-37). Structural experiments using NMR are yielding important results (12,38-41). In general, these studies have provided several conclusions concerning the solution behavior and membrane interactions of CFPs. Amphipathic helical peptides can exist as monomers and aggregates in solution. Monomers are able to interact much more readily with lipid bilayers and micelles. Depending on the peptide to lipid ratios, type of lipid, ionic strength, pH of the solution, and the hydration of the lipid, the peptide will preferentially orient itself either parallel to or perpendicular to the plane of the bilayer. Many CFPs do not require a potential difference across the bilayer to insert spontaneously into the bilayer. Once in the membrane, the helices associate in a time and concentration dependent manner to form the multistate helical bundles. It is these assemblies that conduct the ions across the bilayer. These studies, when considered together, reveal the transmembrane amphipathic helix to be a dynamic structure. The ability to oligomerize in the membrane into stable ring structures, with a central aqueous pore capable of opening and closing, appears to be driven by the asymmetric alignment of hydrophilic and hydrophobic amino acid residues that seem to obey a unique set of rules.
Putative channel forming segments from large channel proteins behave much like the small naturally occurring CFPs described above. They spontaneously insert into bilayers and self-assemble into an ion-conducting structure, presumably comprised of a parallel array of .alpha.-helices. These structures also retain biological activities reminiscent of the native proteins they were modeled after (10, 42-47). These structures are reasonable models for exploring both the oligomerization of transmembrane segments and for defining the molecular events that give rise to channel activity. The beauty of this system emanates from the appearance of a measurable activity that arises from the assembly of an amphipathic transmembrane helix. The activity allows measurement of the effects of amino acid substitutions on either the size of the assemblies or the contribution of the residues to ion selectivity or translocation. The number of helices per channel can be precisely controlled, thus preventing multiple oligomerization states, by tethering the helical segments to a peptide backbone during synthesis. The small size of these assemblies makes them ideally suited for NMR structural studies using either detergent micelle solution NMR or oriented bilayer solid-state NMR.
Pharmacological studies have been a relatively recent addition to the single channel analysis of these model CFP channels. Using a four helix bundle CFP derived from the human L-type dihydropyridine sensitive Ca.sup.2+ channel, the binding of a local anaesthetic as well as a number of calcium channel blockers with binding affinities on the order of those observed for the full length calcium channel protein have been observed (42,43,48). This avenue of investigation adds a sensitive method of discriminating between channels that truly mimic their parent structures as opposed to those that might produce non-discriminating ionic pores. Once the three dimensional structure for one of the synthetic channels has been solved, rational drug design of both channel agonists and antagonists may be attempted using these coordinates.
Membrane proteins are generally acknowledged to be the most difficult class of proteins for detailed structural analysis. The studies presented above clearly demonstrate the utility of working with channel forming peptides, as model systems, to study events involved in peptide association with the bilayer, insertion into membranes, and assembly into oligomers. The amphipathic helix is a suitable structural motif for the pore of channel proteins that also contributes to the organization, size, function, and stabilization of ionic channels. As an assembled structure these helical bundles can be used to investigate the structure, organization, and function of channels.
Application of synthetic peptides to biological membranes. A synthetic peptide with the sequence of the M2.delta. segment of the nicotinic acetylcholine receptor from Torpedo californica forms ion channels in lipid bilayers that emulate those of authentic acetylcholine receptor ion channels (49). Human erthyrocytes exposed to the synthetic peptide released hemoglobin and K.sup.-. Evidently the peptide molecules self-assembled in the membrane to form trimers and pentamers (49). Extensive evidence indicates that Cl.sup.- secretion drives fluid secretion in Madin-Darby canine kidney (MDCK) cells and in cells cultured from the cystic epithelium of the kidneys of patients with autosomal dominant polycystic kidney disease (APKD), and that a Cl.sup.- channel is involved in fluid secretion (50-54). Indeed there is now extensive data indicating that CFTR is the channel involved in that secretion by ADPKD cells (55-57). Apparently, a net secretion of Cl.sup.- into the lumen of the cysts leads to an increase in water volume in the cysts, ultimately resulting in kidney dysfunction. However, although there is a precedent for the application of synthetic channel-forming peptides to cells, no one previously has used channel-forming peptides to treat symptoms of any disease.
U.S. Pat. No. 5,543,399 describes the purification and lipid reconstitution of CFTR protein and CF therapy making use of that protein. There is no teaching or suggestion in this reference of the use of relatively small, easily prepared pure peptides, and particularly peptides which are fragments of channel-forming proteins.
U.S. Pat. No. 5,368,712 teaches the use of small peptides reconstituted in artificial membranes as diagnostic tools. This patent does not describe any therapeutic applications using such peptides.